Solution containing platelet activation inhibitors for pathogen reducing and storing blood platelets

ABSTRACT

This invention relates to the addition of platelet activation inhibitors to solutions used in the pathogen reduction and subsequent storage of platelets. More particularly, the invention relates to the addition of adenylate cyclase stimulators and phosphodiesterase inhibitors to a platelet pathogen reduction and storage solution.

CROSS REFERENCE To RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional patentapplication No. 60/375,670 filed Apr. 26, 2002.

BACKGROUND

[0002] Contamination of blood supplies with infectious microorganismssuch as HIV, hepatitis and other viruses and bacteria presents a serioushealth hazard for those who must receive transfusions of whole blood oradministration of various blood components such as platelets, red cells,blood plasma, Factor VIII, plasminogen, fibronectin, anti-thrombin III,cryoprecipitate, human plasma protein fraction, albumin, immune serumglobulin, prothrombin, plasma growth hormones, and other componentsisolated from blood plasma. Blood screening procedures which arecurrently available may miss contaminants. Thus, there is a need forsterilization procedures that effectively neutralize all infectiousviruses and other microorganisms but do not damage cellular bloodcomponents, do not degrade desired biological activities of proteins,and preferably do not need to be removed prior to administration of theblood product to the patient.

[0003] One method used to sterilize blood and blood components requiresthe use of photosensitizers, compounds which absorb light of a definedwavelength and transfer the absorbed energy to an energy acceptor.

[0004] It is known in the art to use photosensitizers as a component ofsolutions used to photodecontaminate contaminants which may be in bloodand blood products. U.S. Pat. No. 5,709,991 to Lin et al. teaches theuse of the photosensitizer psoralen for photodecontamination of plateletpreparations and removal of photolysed psoralen afterward. U.S. Pat. No.5,459,030 also issued to Lin teaches a platelet storage solutioncontaining 8-methoxypsoralen for use in a pathogen reduction process ofplatelets. U.S. Pat. Nos. 5,712,085, 5,908,742, 5,955,256, 5,965,349,6,017,691 and 6,251,580 all disclose solutions for use in the pathogenreduction of blood which include psoralen or psoralen derivatives as thephotosensitizer.

[0005] It has been found that platelets which have been treated with aphotosensitizer and light to reduce pathogens which may be present arelikely to become activated during long term storage after such atreatment. In addition to causing aggregation of the platelets, thepathogen reduction process also may yield high GMP-140 expression andlow ESC (extended shape change) response by day 5 of storage, both ofwhich may be indications of cytoskeletal changes in the platelets. Suchchanges may be indications of platelet damage which may have occurred asa result of the storage conditions. It is therefore important to improvethe quality of stored pathogen reduced platelets. It is against thisbackground that the present invention is directed.

SUMMARY OF THE INVENTION

[0006] The invention generally relates to synthetic media for use in thepathogen reduction and subsequent storage of platelets. By the term“synthetic media” the present invention intends to indicate aqueoussolutions (e.g., phosphate buffered, aqueous salt solutions) other thanthose naturally occurring (e.g., plasma, serum, etc.). However, it iscontemplated that such synthetic media may also be used in combinationwith naturally occurring fluids. The platelet pathogen reduction andstorage solution of this invention comprises a fluid containingplatelets, a photosensitizer, and at least one platelet activationinhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0007] FIGS. 1-6 are standard measures used to determine the structuralintegrity, respiratory activity and functionality of stored platelets.These standard measures may also be used to measure the cell quality ofplatelets which have been pathogen reduced and stored over five days insolutions with and without platelets activation inhibitor additives.

[0008]FIG. 1 is a graph of the cell count of pathogen reduced plateletsover 5 days of storage.

[0009]FIG. 2 is a graph showing the changes in pH of pathogen reducedplatelets over 5 days of storage.

[0010]FIG. 3 shows glucose consumption of pathogen reduced plateletsover 5 days of storage.

[0011]FIG. 4 shows lactose production of pathogen reduced platelets overa 5 day storage period.

[0012]FIG. 5 is a measure of the % recovery from HSR of pathogen reducedplatelets over 5 days of storage.

[0013]FIG. 6 shows expression of P-selectin by platelets stored over 5days.

[0014]FIG. 7 shows VEGF release by pathogen reduced platelets over fivedays of storage.

[0015]FIG. 8 shows RANTES release by pathogen reduced platelets overfive days of storage.

[0016]FIG. 9 is a graph of the upregulation of cAMP in pathogen reducedplatelets over five days of storage.

[0017]FIG. 10 shows an embodiment of this invention using a bag tocontain the fluid containing platelets being treated with thephotosensitizer and platelet activation inhibitor additives, and ashaker table to agitate the fluid while exposing to photoradiation froma light source.

DETAILED DESCRIPTION

[0018] This invention provides a synthetic aqueous solution forprolonging the preservation of human blood platelets either during orafter a pathogen reduction procedure. The method uses inhibitors ofplatelet activation which help platelets retain their functionalintegrity during prolonged storage after a pathogen reduction procedure,or during the pathogen reduction procedure itself. This is accomplishedby inhibiting normal platelet function, so as to help keep plateletsfrom becoming activated during the pathogen reduction process orafterwards during storage.

[0019] Platelet activation during a pathogen reduction procedure andsubsequent storage is undesirable. However, treated platelets mustretain the ability to function normally and become activated in responseto a stimulus when they are taken out of long term storage andtransfused into a patient.

[0020] There are three parameters of platelet activity that are commonlyused to measure whether pathogen reduced platelets have retained theirfunctional ability after storage. These tests can also be used tomeasure platelet function after a pathogen reduction procedure. Plateletnumber, hypotonic stress response, and agonist-induced plateletaggregation are some commonly used measures which may be used to measureplatelet cell quality during and after a pathogen reduction procedure.

[0021] Hypotonic stress response is an assay used to determine ifplatelets have retained metabolic viability. This assay is a photometricmeasurement of the platelets' ability to overcome the addition of ahypotonic solution. This activity reflects cell function (i.e. afunctional membrane water pump) and is indicative of platelet recoveryfollowing storage. Hypotonic stress response has been demonstrated to bean important indicator of platelets' ability to survive in circulationfollowing transfusion. Consequently, hypotonic stress responserepresents a crucial parameter for evaluating platelet biochemistryfollowing storage.

[0022] Potential for aggregation is another feature that demonstrateswhether blood platelets have maintained their functional integrityduring storage. This potential is measured using agonist-inducedaggregation, where the aggregation or clumping of platelets is theresponse. The agonists, ADP and collagen, are used to induce aggregationto determine if platelets have retained their ability to aggregate inresponse to a stimulus. In addition, when performing aggregationresponses one can detect the presence of spontaneous aggregation, thatis the platelets adhering to each other without the addition of anagonist. The occurrence of spontaneous aggregation has been correlatedwith removal of platelets from the circulation and hence have shortsurvival times in a transfusion recipient.

[0023] The platelet activation inhibitor system of this invention isbased on the addition to a pathogen reduction and/or storage solution ofspecific second messenger effectors which help stabilize the plateletsduring the pathogen reduction process as well as during storage to helpthe pathogen reduced platelets remain viable and retain their functionalactivity.

[0024] Many synthetic solutions to prevent the activation of plateletsduring storage are known. For example, U.S. Pat. No. 5,919,614 toLivesay et al. discloses a solution for the long term storage ofplatelets at reduced temperatures. The storage solution of this patentuses a platelet inhibitor system to keep platelets from activatingduring storage. U.S. Pat. No. 4,994,367 to Bode at al. also discloses asolution for the long-term storage of platelets which includes plateletactivation inhibitors. Neither of these storage solutions are directedtowards a solution for pathogen reduction and subsequent storage ofplatelets.

[0025] The term “platelet activator inhibitor” as used herein includesadenylate cyclase stimulators and phosphodiesterase inhibitors.

[0026] The addition of prostaglandins to platelet storage solutions hasbeen suggested as a potential resolution to the problem of plateletactivation and subsequent aggregation during storage. Prostaglandinshelp to inhibit platelet activation through stimulation of adenylatecyclase, which produces a rapid (but transient) increase inintracellular cAMP (adenosine 3′, 5′-cyclic phosphate). In platelets,cAMP helps block activation pathways and cytoskeletal changes whichoccur upon platelet activation. cAMP (alone and in concert with cGMP)has been implicated in preventing the up regulation of proteins whichhelp to trigger platelet activation, such as p-selectin and gp2bIIa.

[0027] In the present invention, the adenylate cyclase stimulator isadded to the pathogen reduction and/or storage solution in an amounteffective to increase the production of cAMP in the blood platelets andprevent activation of the irradiated platelets during the pathogenreduction process and storage. Exemplary adenylate cyclase stimulatorswhich may be used in the present invention to increase intracellularcAMP and inhibit platelet activation include Prostaglandin E1 (PGE₁) andforskolin. These adenylate cyclase stimulators may be used alone, incombination with other adenylate cyclase stimulators or with otherplatelet activation inhibitors. PGE₁ may be used at a finalconcentration between about 100 nM and 500 nM, preferably about 150 nM.Forskolin may be used at a final concentration between about 1 μM andabout 100 μM, preferably about 3 μM.

[0028] Also included in the pathogen reduction/storage solution of thisinvention are phosphodiesterase inhibitors. Phosphodiesterase inhibitorshelp potentiate the inhibitory effects of prostaglandins, furtherincreasing intracellular cAMP, and preventing platelet activation duringstorage. Compounds which may be used in the present invention includemethylxanthines such as theophylline, caffeine, xanthine, theobromine,aminophylline, oxtriphylline, dyphylline, pentoxifylline,isobutulmethylxanthine, dipyramole, and papaverine. Derivatives ofmethylxanthine may also be used. The above phosphodiesterase inhibitorsmay be used alone, in combination with other phosphodiesteraseinhibitors or with other platelet inhibitors. Exemplaryphosphodiesterase inhibitors which may be used in the present inventioninclude theophylline and caffeine. Theophylline may be used at a finalconcentration of between about 0.90 mM and 2.2 mM, preferably about 0.95mM. Caffeine may be used at a final concentration between about 0.90 mMand 2.2 mM, also preferably about 0.95 mM.

[0029] The amount of platelet activation inhibitor additives to be mixedwith the platelets will be an amount sufficient to adequately maintainthe intracellular cAMP levels in platelets so as to prevent plateletsfrom becoming activated, but not enough to be toxic to the platelets.The platelet activation inhibitor additives may be added directly to theplatelets, or may be added in a pre-mixed aqueous solution, for examplewater, storage buffer or a suspension solution.

[0030] The pathogen reduction componds useful in this invention includeany photosensitizers known to the art to be useful for inactivatingmicroorganisms or other infectious particles. A “photosensitizer” isdefined as any compound which absorbs radiation at one or more definedwavelengths and subsequently utilizes the absorbed energy to carry out achemical process. Examples of such photosensitizers include porphyrins,psoralens, dyes such as neutral red, methylene blue, acridine,toluidines, flavine (acriflavine hydrochloride) and phenothiazinederivatives, coumarins, quinolones, quinones, and anthroquinones.Photosensitizers of this invention may include compounds whichpreferentially adsorb to nucleic acids, thus focusing their photodynamiceffect upon microorganisms and viruses with little or no effect uponaccompanying non-nucleated cells or proteins. Other photosensitizers arealso useful in this invention, such as those using singletoxygen-dependent mechanisms.

[0031] Most preferred are endogenous photosensitizers. The term“endogenous” means naturally found in a human or mammalian body, eitheras a result of synthesis by the body or because of ingestion as anessential foodstuff (e.g. vitamins) or formation of metabolites and/orbyproducts in vivo. Examples of such endogenous photosensitizers arealloxazines such as 7,8-dimethyl-10-ribityl isoalloxazine (riboflavin),7,8,10-trimethylisoalloxazine (lumiflavin), 7,8-dimethylalloxazine(lumichrome), isoalloxazine-adenine dinucleotide (flavine adeninedinucleotide [FAD]), alloxazine mononucleotide (also known as flavinemononucleotide [FMN] and riboflavine-5-phosphate), vitamin Ks, vitaminL, their metabolites and precursors, and napththoquinones, naphthalenes,naphthols and their derivatives having planar molecular conformations.The term “alloxazine” includes isoalloxazines. Endogenously-basedderivative photosensitizers include synthetically derived analogs andhomologs of endogenous photosensitizers which may have or lack lower(1-5) alkyl or halogen substituents of the photosensitizers from whichthey are derived, and which preserve the function and substantialnon-toxicity thereof. Such endogenously-based derivativephotosensitizers which may be used in this invention are disclosed inU.S. Pat. No. 6,268,120 to Platz et al., and discloses alloxazinederivatives which may also be used to inactivate microorganismscontained in blood or blood components. This patent is incorporated byreference into the present invention to the amount not inconsistent.

[0032] When endogenous photosensitizers are used, particularly when suchphotosensitizers are not inherently toxic or do not yield toxicphotoproducts after photoradiation, no removal or purification step isrequired after decontamination, and the treated product can be directlyadministered to a patient by any methods known in the art. Preferredendogenous photosensitizers are:

[0033] The method of this invention requires mixing the pathogenreduction compound and platelet activation inhibitors with the fluidcontaining platelets. Mixing may be done by simply adding the pathogenreduction compound and platelet activation inhibitors or a solutioncontaining the pathogen reduction compound and platelet activationinhibitors directly to the fluid to be pathogen reduced. Plateletactivation inhibitors may be added to the platelets separately from thepathogen reduction compound or they can be added together.

[0034] The fluid containing at least platelets and pathogen reductioncompound and platelet activation inhibitors is exposed to photoradiationfor a time sufficient to reduce any pathogens which may be contained inthe fluid. The wavelength used will depend on the type of pathogenreduction compound selected and the type of blood component beingpathogen reduced. For platelets, the light source may provide light ofabout 270 nm to about 700 nm, and more preferably about 308 nm to about320 nm.

[0035] The light source may be a simple lamp, or may consist of multiplelamps radiating at different wavelengths. The photoradiation sourceshould be capable of delivering from about 1 j/cm² to at least 120J/cm^(2.)

[0036] In one embodiment, the platelets to be decontaminated to which apathogen reduction compound and at least one platelet activationinhibitor has been added is flowed past a photoradiation source, and theflow of the material generally provides sufficient turbulence todistribute the pathogen reduction compound and platelet activationinhibitor throughout the fluid to be pathogen reduced. A separate mixingstep may optionally be added.

[0037] In another embodiment, the fluid, pathogen reduction compound andplatelet activation inhibitor/s are placed in a photopermeable containerand irradiated in batch mode, preferably while agitating the containerto fully distribute the pathogen reduction compound throughout the fluidand expose all the fluid to the radiation. Platelet activationinhibitors may be added to the fluid containing at least platelets andpathogen reduction compound either before the pathogen reductionprocedure as describe above, or after the procedure. In this embodiment,the photopermeable container is preferably a blood bag made oftransparent or semitransparent plastic, and the agitating means ispreferably a mechanism for shaking the container in multiple planes.

[0038]FIG. 10 depicts an embodiment of this invention in which a fluidcontaining platelets to be decontaminated is placed in a bag 284equipped with an inlet port 282, through which photosensitizer andplatelet activation inhibitors 290 may be added from flask 286 via pourspout 288. Shaker table 280 is activated to agitate the bag 284 to mixthe fluid to be decontaminated, the photosensitizer, and the plateletactivation inhibitors together while photoradiation source 260 isactivated to irradiate the fluid and photosensitizer in bag 284. Thephotosensitizer and/or platelet activation inhibitors may be added tothe container in powdered or liquid form, or alternatively, the bag 284can be provided prepackaged to contain photosensitizer and/or plateletactivation inhibitors and the platelets may thereafter be added to thebag. The platelet activation inhibitors may also be added to bag 284through a sterile barrier filter (not shown) connected to inlet port282.

[0039] The amount of photosensitizer to be mixed with the platelets willbe an amount sufficient to adequately inactivate the reproductiveability of a pathogen. Preferably the photosensitizer is used in aconcentration of at least about 1 μM up to the solubility of thephotosensitizer in the fluid. For 7,8-dimethyl-10-ribityl isoalloxazinea concentration range between about 1 μM and about 160 μM is preferred,preferably about 50 μM. The photosensitizer may be added directly to theplatelets, or may be added in a pre-mixed aqueous solution, for examplewater, storage buffer or a suspension solution.

[0040] Additives additional to platelet inactivation inhibitor additivessuch as the glycolytic inhibitor 2-deoxy-D-glucose may also be used withthe platelet pathogen reduction/storage solution of this invention. Inplatelets, 2-deoxy-D-glucose slows down the rate of glycolysis bycompeting with glucose for enzymes utilized in the glycolysis pathway.2-deoxy-D-glucose is phosphorylated by the same enzymes whichphosphorylate glucose, but at a slower rate than that of glucosephosphorylation. Such competitive binding slows the rate of glucosebreakdown by the cell and consequently slows the rate of lactic acidproduction by platelets during storage. Such additives may helpcontribute to platelet viability during and after a pathogen reductionprocedure. 2-deoxy-D-glucose may be added to the pathogenreduction/storage solution in a concentration of about 10 mM.

[0041] Quenchers may also be added to the fluid to make the process moreefficient and selective. Such quenchers include antioxidants or otheragents to prevent damage to desired fluid components or to improve therate of reduction of pathogens and are exemplified by adenine,histidine, cysteine, tyrosine, tryptophan, ascorbate,N-acetyl-L-cysteine, propyl gallate, glutathione,mercaptopropionylglycine, dithiothreotol, nicotinamide, BHT, BHA,lysine, serine, methionine, glucose, mannitol, vitamin E, trolox,alpha-tocopheral acetate and various derivatives, glycerol, and mixturesthereof. Quenchers may be added to the pathogen reduction/storagesolution in an amount necessary to prevent damage to the platelets.

[0042] Pathogen reduction methods as described above may be designed asstand-alone units, or may be incorporated into existing apparatusesknown to the art for reducing pathogens in blood or blood components.The process is further described in U.S. Pat. Nos. 6,277,337 and6,258,577 issued to Goodrich et al., which are incorporated by referenceherein in their entirety to the amount not inconsistent.

EXAMPLES Example 1

[0043] Example 1 shows the results of commonly used measures of plateletcell quality which may be used to measure the effects of plateletactivation inhibitors on platelets during pathogen reduction and/orsubsequent storage. These may be indirect measures of the increase inintracellular cAMP in pathogen reduced and stored platelets.

[0044] FIGS. 1-5 are graphs showing standard measures of platelet cellquality during 5 days of storage. Platelets were separated from wholeblood and collected using a blood collection device such as the COBESpectra™ or TRIMA® apheresis systems (available from Gambro BCT, Inc.,Lakewood, Colo., USA). However, it should be noted that any device knownin the art for separating blood into components may be used to collectplatelets without departing from the spirit ad scope of the presentinvention.

[0045] The platelet activation inhibitors prostaglandin E1 (PGE₁) andtheophylline (Theo) were added either alone or in combination to asolution containing a final concentration of 50 μM riboflavin and salineto make a solution which could be used as a platelet pathogen reductionsolution. The resulting pathogen reduction solution was then added tothe platelets and incubated for 60 minutes. The pathogen reductionsolution and platelets were then irradiated at 12 J/cm². The irradiatedplatelets were stored for 5 days under standard platelet storageconditions at 22° C., and platelet cell quality was determined.

[0046] Untreated, (or control) platelets (designated Un-Tx) wereincubated with riboflavin and irradiated, but platelet activationinhibitors were not added. Platelet activation inhibitors were added toplatelets either before irradiation (designated pre-Tx) or afterirradiation (designated post-Tx). The addition of the plateletactivation inhibitors prior to irradiation is designated in the graphlegend as PGE₁+Theo., pre-Tx. The addition of the platelet activationinhibitors subsequent to irradiation is designated in the graph legendas PGE₁+Theo., post-Tx. Theophylline was added at a final concentrationof 0.95 mM, and PGE₁ was added at a final concentration of 150 mM.

[0047] Treated platelets are defined as irradiated platelets to whichplatelet activation inhibitors were added. The term “treated platelets”encompasses platelets which were irradiated in the presence of aphotosensitizer first followed by the addition of activation inhibitors,as well as platelets to which activation inhibitors were first addedfollowed by irradiation.

[0048] Platelet cell count, pH, glucose consumption, lactate production,and hypotonic stress response (HSR) were measured to determineindirectly the effect platelet activation inhibitors had on cell qualityafter the pathogen reduction process and after storage.

[0049]FIG. 1 is a graph of the cell count of pathogen reduced plateletsover 5 days of storage.

[0050] The addition of activation inhibitors to platelets beforeirradiation substantially prevents platelets from aggregating togetherfollowing treatment. If platelets become activated and aggregatetogether, the number of cells which can be counted in the fluid willdecrease. Although the starting numbers for both treated and untreatedcells are the same at day 0, by day 1, the platelets in solution whichdid not contain additional platelet activation inhibitor additives showdecreased cell numbers, most likely due to platelet clumping (andconsequently platelet activation). The addition of platelet activationinhibitors to platelets before exposure to irradiation appears toprevent platelet clumping to a greater extent than adding plateletactivation inhibitors post-illumination.

[0051]FIG. 2 is a graph showing changes in pH of pathogen reducedplatelets over 5 days of storage.

[0052] The addition of platelet activation inhibitors both before andafter illumination appears to help maintain the pH of the storedplatelets as compared to untreated platelets. Maintenance of pH byplatelets over time is an indirect measure of platelet cell quality. Ifthe pH of the storage solution drops, platelets have a higher likelihoodof becoming activated during the storage process.

[0053]FIG. 3 shows glucose consumption of pathogen reduced plateletsover a 5 day storage period.

[0054] Glucose is broken down by platelets to produce cellular energyvia the glycolytic pathway. To become activated, platelets need toconsume greater amounts of energy than in a non-activated state.Therefore, activated platelets consume glucose at a higher rate thannon-activated platelets. With the addition of platelet activationinhibitors, platelets remain in a non-activated state, and thereforeglucose is consumed at a slower rate by platelets treated with plateletactivation inhibitor additives as compared to untreated platelets.

[0055]FIG. 4 shows lactate production by platelets during storage.

[0056] For every molecule of glucose consumed by a metabolizingplatelet, two molecules of lactic acid are produced. If platelets areactivated, they consume more glucose and consequently produce morelactic acid. Lactic acid buildup within cells causes the pH of thesolution to drop. Such a drop in pH may cause decreased cell qualityduring storage, and may further platelet activation. As shown in FIG. 4,with the addition of platelet activation inhibitor additives lactic acid(or lactate) is produced by platelets at a slower rate compared tountreated platelets.

[0057]FIG. 5 is a measure of the % reversal from exposure to a hypotonicstress (HSR) of pathogen reduced platelets during 5 days of storage.

[0058] As described above, the ability of platelets to overcome theaddition of a hypotonic solution is one measure of platelet cell qualityafter storage. The addition of activation inhibitor additives toplatelets pre and post irradiation appears to significantly contributeto the treated platelets ability to show a reversable hypotonic stressresponse after 5 days of storage compared to untreated platelets.

Example 2

[0059] Other markers of platelet activation after pathogen reduction andstorage were also measured, such as cytokine release and cell surfacemarkers of platelet activation. Such markers are another way ofmeasuring the suitability for transfusion purposes of pathogen reducedplatelets.

[0060] Platelet Surface Antigens

[0061]FIG. 6 shows the expression of GMP-140 on the surface of plateletsstored for 5 days. GMP-140 is a measure of p-selectin which is aplatelet surface protein upregulated upon platelet aggregation.Treatment of platelets with activation inhibitor additives PGE₁ andtheophylline in combination before and after a pathogen reductionprocedure helps to slow the rate of platelet activation during storageas compared to untreated cells.

[0062] Cytokine Measurements

[0063] The supernatent of treated and untreated platelets were assayedto determine levels of RANTES (regulated upon activation, normal T-cellexpressed and secreted) and VEGF (human vascular endothelial growthfactor) produced by treated and untreated platelets. Both cytokines werequantified by a commercially available ELISA (available from R & DSystems, Wiesbaden-Nordenstadt, Germany) according to manufacturer'sinstructions.

[0064] The chemokine RANTES is known to be released from activatedplatelets. VEGF has also been reported to be released upon plateletactivation. The presence of enhanced levels of VEGF and RANTES could beadditional markers used to determine the transfusion efficacy ofplatelets which have undergone a pathogen reduction procedure in thepresence of platelet activation inhibitors.

[0065]FIG. 7 measures VEGF released by pathogen reduced platelets overfive days of storage in the presence or absence of platelet activatorinhibitor additives. Platelets were irradiated with 12 J/cm² light inthe presence of riboflavin either with or without the plateletactivation inhibitor additives forskolin and caffeine. 3 μM forskolinand 1.9 mM caffeine were added to the platelets. As shown, the presenceof forskolin and caffeine substantially reduced the release of VEGF byplatelets over five days of storage.

[0066]FIG. 8 is a measure of RANTES released by pathogen reduced andstored platelets. FIG. 8 compares the amount of RANTES released byplatelets which have been irradiated at 12 J/cm² in the presence ofriboflavin, which were than stored over five days in a solution whichcontined no additional platelet activation inhibitor additives ascompared to platelets stored in a solution containing 0.95 mM caffeineand 3 mM forskolin. As shown, the combination of an adenylate cyclasestimulator (forskolin) and a phosphodiesterase inhibitor (caffeine) to aplatelet storage solution results in a decrease in the amount of RANTESreleased by platelets as compared to platelets stored in a solutionwhich does not contain additional platelet activation inhibitoradditives.

[0067] cAMP

[0068] The effects of platelet activation inhibitors on the upregulationof cAMP by platelets which were pathogen reduced and stored can bemeasured directly using commercially available cAMP detection kits. Onesuch kit which may be used is a fluorescent assay for cAMP availablefrom Cayman Chemical (Ann Arbor, Mich., USA).

[0069]FIG. 9 is a direct measurement of cAMP produced by platelets overfive days of storage. cAMP was measured according to manufacturer'sinstructions. Platelets which were pathogen reduced and stored insolutions containing 30 μM forskolin and 1.0 mM caffeine continued tomaintain sufficient cAMP levels, thus presumably not becoming activatedduring storage.

[0070] In describing the chemicals which have shown utility as plateletactivation inhibitors, it must be understood that the actual chemicalsmentioned together with functionally equivalent materials are intendedto be within the scope of this invention. Chemicals that are known toapplicants to have known or demonstrated utility as platelet activationinhibitors have been specifically set forth in the instant application.However, it is intended that the scope of the application be extended toother functionally effective chemicals, both existing chemicals andchemicals yet to be discovered.

[0071] Platelet activation inhibitors containing adenylate cyclasestimulators and phosphodiesterase inhibitors may be added either aloneor in combination to a solution containing platelets to be pathogenreduced. PGE₁, forskolin, theophylline and caffeine either alone or incombination may be added.

[0072] It is also understood that other platelet additives such as2-deoxy-D-glucose may also be added to improve platelet storageconditions. Quenchers or a combination of quenchers as set forth abovemay also be used.

[0073] In one embodiment, the present invention contemplates an aqueoussynthetic pathogen reduction solution comprising saline, aphotosensitizer and at least one platelet activation inhibitor. Water ora physiological buffer may replace saline in the pathogen reductionsolution.

Example 3

[0074] In an alternative embodiment, the platelet activation inhibitoradditives may be added to any commercially available synthetic plateletstorage solutions after the pathogen reduction process, to aid in longterm platelet storage. The solutions listed below contain thephotosensitizer isoalloxazine, but any pathogen reduction compound maybe used.

[0075] This example compares novel blood component additive solutionsfor addition to platelets separated from whole blood. Six commerciallyavailable solutions were used: PAS II, PSMI-pH, PlasmaLyte A, SetoSol,PAS III, and PAS. To each known solution was added an effective amountof an endogenous photosensitizer, 7,8-dimethyl-10-ribityl isoalloxazineas well as an effective amount of at least one platelet activationinhibitor such as PGE₁ and/or theophylline or other methylxanthine. For7,8-dimethyl-10-ribityl isoalloxazine a final or working concentrationrange between about 1 μM and about 160 μM is preferred, preferably about50 μM. The composition of each solution is shown in Table 3a below, andvaries in the amount of blood component additives present. The bloodadditive components may be in a physiological solution, as well as a drymedium adapted to be mixed with a solvent, including tablet, pill orcapsule form. TABLE 3a Blood Component Platelet Storage SolutionAdditive PSS 1 PSS 2 PSS 3 PSS 4 PSS 5 PSS 6 KCl (mM) 5.0 5.0 5.0 5.1CaCl₂ (mM) 1.7 MgCl₂ (mM) 3.0 3.0 MgSO₄ (mM) 0.8 sodium 10.0 23.0 23.017.0 15.2 12.3 citrate (mM) citric 2.7 acid (mM) NaHCO₃ (mM) 35.0Na₂HPO₄ 25.0 25.0 2.1 28.0 (mM) sodium 30.0 27.0 23.0 42.0 acetate (mM)sodium 23.0 gluconate (mM) glucose (mM) 23.5 38.5 maltose (mM) 28.87,8-dimethyl 50.0 50.0 50.0 50.0 50.0 50.0 10-ribityl isoalloxazine (μM)platelet variable variable variable variable variable variableactivation inhibitor

[0076] In Example 3, the platelet storage solution PSS 1 (also known asPAS II) comprises a physiological saline solution, tri-sodium citrate ata concentration of approximately about 10 mM, sodium acetate at aconcentration of approximately about 30 mM, 7, 8-dimethyl-10-ribitylisoalloxizine at a concentration of about 50 μM and an effective amountof at least one platelet activation inhibitor.

[0077] The platelet storage solution PSS 2 (also known as PSMI-pH)comprises a physiological saline solution, potassium chloride at aconcentration of approximately about 5 mM, tri-sodium citrate at aconcentration of approximately about 23 mM, a mixture of monosodiumphosphate and dibasic sodium phosphate at a concentration ofapproximately about 25 mM, 7, 8-dimethyl-10-ribityl isoalloxizine at aconcentration of about 50 μM and an effective amount of at least oneplatelet activation inhibitor.

[0078] The platelet storage solution PSS 3 (also known as PlasmaLyte A)comprises a physiological saline solution, potassium chloride at aconcentration of approximately about 5 mM, magnesium chloride at aconcentration of approximately about 3 mM, tri-sodium citrate at aconcentration of approximately about 23 mM, sodium acetate at aconcentration of approximately about 27 mM, sodium gluconate at aconcentration of approximately about 23 mM, 7, 8-dimethyl-10-ribitylisoalloxizine at a concentration of about 50 μM and an effective amountof at least one platelet activation inhibitor.

[0079] Platelet storage solution PSS 4 (also known as SetoSol) comprisesa physiological saline solution, potassium chloride at a concentrationof approximately about 5 mM, magnesium chloride at a concentration ofapproximately about 3 mM, tri-sodium citrate at a concentration ofapproximately about 17 mM, sodium phosphate at a concentration ofapproximately about 25 mM, sodium acetate at a concentration ofapproximately about 23 mM, glucose at a concentration of approximatelyabout 23.5 mM, maltose at a concentration of approximately about 28.8mM, 7,8-dimethyl-10-ribityl isoalloxizine at a concentration of about 50μM and an effective amount of at least one platelet activationinhibitor.

[0080] Platelet storage solution PSS 5 (also known as PAS III) comprisesa physiological saline solution, potassium chloride at a concentrationof approximately about 5.1 mM, calcium chloride at a concentration ofapproximately about 1.7 mM, magnesium sulfate at a concentration ofapproximately about 0.8 mM, tri-sodium citrate at a concentration ofapproximately about 15.2 mM, citric acid at a concentration ofapproximately about 2.7 mM, sodium bicarbonate at a concentration ofapproximately about 35 mM, sodium phosphate at a concentration ofapproximately about 2.1 mM, glucose at a concentration of approximatelyabout 38.5 mM, 7,8-dimethyl-10-ribityl isoalloxizine at a concentrationof about 50 μM and an effective amount of at least one plateletactivation inhibitor.

[0081] Platelet storage solution PSS 6 (also known as PAS) comprises aphysiological saline solution, tri-sodium citrate at a concentration ofapproximately about 12.3 mM, sodium phosphate at a concentration ofapproximately about 28 mM, sodium acetate at a concentration ofapproximately about 42 mM, 7,8-dimethyl-10-ribityl isoalloxizine at aconcentration of about 50 μM and an effective amount of at least oneplatelet activation inhibitor.

[0082] In an aspect of this embodiment, physiologic saline may bereplaced with a solvent comprising water and an effective amount ofsodium chloride.

[0083] The blood additive solution may also comprise any other syntheticadditive solution including an effective amount of7,8-dimethyl-10-ribityl isoalloxazine and a platelet activationinhibitor in a liquid, pill or dry medium form. PSS 7, PSS 8 and PSS 9(set forth in Table 3b below) are further examples of platelet pathogenreduction and/or storage solutions which may be used in the presentinvention. TABLE 3b Platelet Storage Solution Blood Component AdditivePSS 7 PSS 8 PSS 9 NaCl (mM) 115.0 78.3 68.5 potassium cloride (mM) 5.75.0 MgCl₂ (mM) 1.7 1.5 sodium citrate (mM) 10.0 sodium phosphate(monobasic) 6.2 5.4 8.5 sodium phosphate (dibasic) 19.8 24.6 21.5 sodiumacetate (mM) 30.0 34.3 30.0 7,8-dimethyl 10-ribityl isoalloxazine (μM)14.0 variable 14.0 platelet activation inhibitor variable variablevariable

[0084] As described in Table 3b, PSS 7 was prepared in RODI water andsodium chloride at a concentration of approximately 115 mM, sodiumcitrate at a concentration of approximately 10.0 mM, sodium phosphate(monobasic) at a concentration of approximately 6.2 mM, sodium phosphate(dibasic) at a concentration of approximately 19.8 mM, sodium acetate ata concentration of approximately 30.0 mM, 7,8-dimethyl 10-ribitylisoalloxazine at a concentration of approximately 14.0 μM and aneffective amount of at least one platelet activation inhibitor. PSS 7has a pH of 7.2.

[0085] PSS 8 was prepared in RODI water and comprises and sodiumchloride at a concentration of approximately 78.3 mM, potassium chlorideat a concentration of approximately 5.7 mM, magnesium chloride at aconcentration of approximately 1.7 mM, sodium phosphate (monobasic) at aconcentration of approximately 5.4 mM, sodium phosphate (dibasic) at aconcentration of approximately 24.6 mM, sodium acetate at aconcentration of approximately 34.3 mM, a variable concentration of7,8-dimethyl 10-ribityl isoalloxazine and an effective amount of atleast one platelet activation inhibitor. PSS 8 has a pH of 7.4, and anosmolarity of 297 mmol/kg.

[0086] PSS 9 was prepared in RODI water and comprises and sodiumchloride at a concentration of approximately 68.5 mM, potassium chlorideat a concentration of approximately 5.0 mM, magnesium chloride at aconcentration of approximately 1.5 mM, sodium phosphate (monobasic) at aconcentration of approximately 8.5 mM, sodium phosphate (dibasic) at aconcentration of approximately 21.5 mM, sodium acetate at aconcentration of approximately 30.0 mM, 7,8-dimethyl 10-ribitylisoalloxazine at a concentration of approximately 14.0 μM and aneffective amount of at least one platelet activation inhibitor. PSS 9has a pH of 7.2, and an osmolarity of 305 mmol/kg.

[0087] While the compositions and methods of this invention have beendescribed in terms of preferred embodiments, it will be apparent tothose of skill in the art that variations may be applied to theprocesses described herein without departing from the concept, spiritand scope of the invention. All such similar substitutions andmodifications apparent to those skilled in the art are deemed to bewithin the spirit, scope and concept of the invention as it is set outin the following claims.

1. A method for maintaining the suitability for transfusion purposes ofplatelets which may contain pathogens during a pathogen reductionprocess comprising the steps of: adding a pathogen reducing compound tothe platelets; adding to the platelets and pathogen reducing compound atleast one platelet activation inhibitor additive; and exposing theplatelets and pathogen reducing compound and at least one plateletactivation inhibitor additive to light of a sufficient wavelength toreduce any pathogens contained in the platelets.
 2. The method of claim1 wherein the step of adding to the platelets and pathogen reducingcompound at least one platelet activation inhibitor additive occursprior to the exposing step.
 3. The method of claim 1 wherein the step ofadding to the platelets and pathogen reducing compound at least oneplatelet activation inhibitor additive occurs subsequent to the exposingstep.
 4. The method of claim 1 wherein the at least one plateletactivation inhibitor additive is an adenylate cyclase stimulator in anamount effective to increase the production of adenosine 3′, 5′ cyclicphosphate in the platelets.
 5. The method of claim 1 wherein theadenylate cyclase stimulator is selected from the group consisting ofPGE₁ and forskolin.
 6. The method of claim 5 wherein PGE₁ is added at afinal concentration of between about 100 nM and 500 nM.
 7. The method ofclaim 6 wherein PGE₁ is added at a final concentration of 150 nM.
 8. Themethod of claim 5 wherein forskolin is added at a final concentration ofbetween about 1 μM and 100 μM.
 9. The method of claim 8 whereinforskolin is added at a final concentration of about 3 μM.
 10. Themethod of claim 1 wherein the at least one platelet activation inhibitoradditive is a phosphodiesterase inhibitor in an amount effective toreduce the degradation of cAMP in the platelets.
 11. The method of claim10 wherein the phosphodiesterase inhibitor is a methylxanthine.
 12. Themethod of claim 10 wherein the phosphodiesterase inhibitor is selectedfrom the group consisting of xanthine, theophylline, caffeine,theobromine, aminophylline, oxtriphylline, dyphylline, pentoxifylline,isobutulmethylxanthine, dipyramole, and papaverine.
 13. The method ofclaim 11 wherein the methylxanthine is theophylline added at a finalconcentration of between about 0.90 mM and 2.2 mM.
 14. The method ofclaim 13 wherein the theophylline is added at a final concentration ofabout 0.95 mM.
 15. The method of claim 11 wherein the methylxanthine iscaffeine added at a final concentration of between about 0.9 mM and 2.2mM.
 16. The method of claim 15 wherein the caffeine is added at a finalconcentration of about 0.95 mM.
 17. The method of claim 1 wherein thestep of adding to the platelets and pathogen reducing compound at leastone platelet activation inhibitor additive further comprises adding asecond platelet activation inhibitor additive.
 18. The method of claim17 wherein the at least one platelet activation inhibitor additivecomprises an adenylate cyclase stimulator and the second plateletactivation inhibitor additive comprises a phosphodiesterase inhibitor.19. The method of claim 1 wherein the step of adding to the plateletsand pathogen reducing compound at least one platelet activationinhibitor additive further comprises adding a glycolytic inhibitor. 20.The method of claim 19 wherein the glycolytic inhibitor is2-deoxy-D-glucose.
 21. The method of claim 1 wherein the step of addingto the platelets and pathogen reducing compound at least one plateletactivation inhibitor additive further comprises adding at least onequencher.
 22. The method of claim 21 wherein the quencher is selectedfrom the group consisting of adenine, histidine, cysteine, tyrosine,tryptophan, ascorbate, N-acetyl-L-cysteine, propyl gallate, glutathione,mercaptopropionylglycine, dithiothreotol, nicotinamide, BHT, BHA,lysine, serine, methionine, glucose, mannitol, vitamin E, trolox,alpha-tocopheral acetate and various derivatives, glycerol, and mixturesthereof.
 23. The method of claim 1 wherein the pathogen reducingcompound comprises a photosensitizer.
 24. The method of claim 23 whereinthe photosensitizer is an endogenous photosensitizer.
 25. The method ofclaim 24 wherein the endogenous photosensitizer is riboflavin.
 26. Astorage solution for maintaining the cell quality of pathogen reducedplatelets during storage comprising: a pathogen reducing compound; andat least one platelet activation inhibitor additive.
 27. The storagesolution of claim 26 wherein the storage solution may also be used as apathogen reduction solution.
 28. The storage solution of claim 26wherein the at least one platelet activation inhibitor comprises anadenylate cyclase stimulator in an amount effective to increase theproduction of adenosine 3′, 5′ cyclic phosphate in the platelets. 29.The storage solution of claim 28 wherein the adenylate cyclasestimulator is selected from the group consisting of PGE₁ and forskolin.30. The storage solution of claim 29 wherein PGE₁ is added at a finalconcentration of between about 100 nM and 500 nM.
 31. The storagesolution of claim 30 wherein PGE₁ is added at a final concentration of150 nM.
 32. The storage solution of claim 29 wherein forskolin is addedat a final concentration of between about 1 μM and 100 μM.
 33. Thestorage solution of claim 32 wherein forskolin is added at a finalconcentration of about 3 μM.
 34. The storage solution of claim 26wherein the at least one platelet activation inhibitor additive is aphosphodiesterase inhibitor in an amount effective to reduce thedegradation of cAMP in the platelets.
 35. The storage solution of claim34 wherein the phosphodiesterase inhibitor is a methylxanthine.
 36. Thestorage solution of claim 34 wherein the phosphodiesterase inhibitor isselected from the group consisting of xanthine, theophylline, caffeine,theobromine, aminophylline, oxtriphylline, dyphylline, pentoxifylline,isobutulmethylxanthine, dipyramole, and papaverine.
 37. The storagesolution of claim 35 wherein the methylxanthine is theophylline added ata final concentration of between about 0.90 mM and 2.2 mM.
 38. Thestorage solution of claim 37 wherein the theophylline is added at afinal concentration of about 0.95 mM.
 39. The storage solution of claim35 wherein the methylxanthine is caffeine added at a final concentrationof between about 0.90 mM and 2.2 mM.
 40. The storage solution of claim39 wherein the caffeine is added at a final concentration of about 0.95mM.
 41. The storage solution of claim 26 wherein the at least oneplatelet activation inhibitor additive further comprises a secondplatelet activation inhibitor additive.
 42. The storage solution ofclaim 41 wherein the at least one platelet activation inhibitor additivecomprises an adenylate cyclase stimulator and the second plateletactivation inhibitor additive comprises a phosphodiesterase inhibitor.43. The storage solution of claim 26 further comprising a glycolyticinhibitor.
 44. The storage solution of claim 43 wherein the glycolyticinhibitor is 2-deoxy-D-glucose.
 45. The storage solution of claim 26further comprising at least one quencher.
 46. The storage solution ofclaim 45 wherein the quencher is selected from the group consisting ofadenine, histidine, cysteine, tyrosine, tryptophan, ascorbate,N-acetyl-L-cysteine, propyl gallate, glutathione,mercaptopropionylglycine, dithiothreotol, nicotinamide, BHT, BHA,lysine, serine, methionine, glucose, mannitol, vitamin E, trolox,alpha-tocopheral acetate and various derivatives, glycerol, and mixturesthereof.
 47. The storage solution of claim 26 wherein the pathogenreducing compound comprises a photosensitizer.
 48. The storage solutionof claim 47 wherein the photosensitizer is an endogenousphotosensitizer.
 49. The storage solution of claim 48 wherein theendogenous photosensitizer is riboflavin.
 50. The storage solution ofclaim 26 further comprising a solvent.
 51. The storage solution of claim50 wherein the solvent is selected from the group consisting of PSS1,PSS2, PSS3, PSS4, PSS5, PSS6, PSS7, PSS8 and PSS9.
 52. The storagesolution of claim 50 wherein the solvent is selected from the groupconsisting of saline and water.
 53. A fluid for transfusing into arecipient comprising: platelets; a pathogen reduction compound; and oneor more platelet activation inhibitor additives.